Zebrafish embryos time-course, 5'UTR nup43

This Zenodo file contains data for the 5′ UTR-nup43-sfGFP mRNA reporter. Data consists of raw microscopy images of embryos, as well as csv files with quantification of relative sfGFP expression data (normalized to a dextran dye control) and png files depicting ROIs used for quantification presented in Figure 2 of Reimão-Pinto et al., Dev Cell, 2024.

Embryos were injected with 1 nL of an injection mix directly into the cell at the 1-cell stage with a microinjection needle (Sutter Instruments) and allowed to develop in standard conditions. For single-reporter injections, 40pg 5′ UTR-sfGFP test reporter were co-injected with 2 ng fluorescent red dextran dye (D1868, Invitrogen) as injection control per embryo. Embryos were collected at the desired developmental stage and placed on a custom-made agarose mold with squared indents for placing and aligning the embryos. For fluorescence intensity quantification, zebrafish embryo images were acquired using an upright ZEISS Axiozoom coupled to an Axiocam 503 color/mono digital camera (14-bit depth) in black & white color mode with fixed laser power (red laser power 85% and 300 ms exposure; green laser power 65% and 300 ms exposure), fixed zoom and fixed exposure time for red mRF12 (590/612) and green AF488 (493/517) channels. Two rounds of single-reporter injections (embryos from two different clutches) were performed for fluorescence intensity quantifications.

Images were quantified using Fiji (Image J) using a macro for automated thresholding and channel fluorescence intensity measurement. For each image, the script automatically segments the image based on red channel intensity (control dextran dye) using FIJI’s auto thresholding tool (RenyiEntropy) and outputs csv files with mean fluorescence intensities of red and green channels for that region of interest (and a small invariant region for background correction). The mean fluorescence values outputted were then used for calculating normalized mean intensity ratios (sfGFP/dextran). A total of 25 embryos per injection round were quantified, for a total of 50 embryos per time-point, per reporter. This excel contains normalized fluorescence intensity values calculated. More embryos were quantified than the ones included in the analysis (due to unequal number of embryos imaged per experiment, we decided to consider a fixed number of 25 per reporter injection). Some embryos were not considered for normalized intensity calculations due to improper automatic segmentation (e.g. due to the presence of a background dirt spot that is recognized as "signal"), due to precipitation of the dextran dye in the embryo's chorion or due to improper embryo development resulting from injury from injection. All images acquired are available for inspection. The FIJI macro for automated segmentation and ROI intensity measurements are also provided in the Mendeley Repository associated to the study.